Taq polymerase sequences useful for incorporating analogs of nucleoside triphosphates

ABSTRACT

This patent applications concerns compositions of matter that are DNA polymerases, where those polymerases have had one or more of their amino acids replaced at sites chosen by an analysis of patterns of conservation and replacement within homologous protein sequences. Disclosed here are sites within Family A DNA polymerases where amino acid replacement creates polymerases having utility, in an example where DNA nucleotides are incorporated having modified or unnatural nucleobases, and/or nucleotides whose sugar is unnatural or derivatized, including 3′-O-amino-2′-deoxyribonucleoside triphosphates. The claimed compositions include polymerases that hold amino acid replacements at claimed sites in Taq polymerase, and are prepared by site-directed mutagenesis that modifies a gene encoding a parent Taq gene (natural or already mutated) to change the codon encoding the amino acid at the claimed site, giving a variant gene that encodes a Taq polymerase protein that has a different amino acid at the claimed site.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

The Federal government may have certain rights through its sponsorshipof this research under NIH grant R21HG003581

SEQUENCE LISTING

A sequence listing is appended, on a compact list and printable form (31pp). Both forms are identical

FIELD

This invention relates to the field of biotechnology, more specificallythat part of this field that concerns the enzymatic synthesis of DNAusing DNA polymerases, and most specifically the enzymatic synthesis ofDNA that involves the incorporation of analogs of natural nucleosidetriphosphates

BACKGROUND

The ability to sequence and re-sequence (a term that describes thesequencing of a new genome while making reference to the genome of aclosely related organism, generally of the same species)deoxyribonucleic acid (DNA) has the potential for revolutionizingbiology and medicine. The ability to re-sequence segments of the genomeof individual humans will enable the personalization of medicine, as thegenetic differences between individuals carries information about howthose individuals will respond differently to similar treatments[Ros00].

Most DNA sequencing is done today using capillary array DNA sequencersthat detect fluorescent dyes appended to the 5- or 7-positions ofpyrimidine or 7-deazapurine nucleobases attached to dideoxynucleotideanalogs [Smi86][Ju95][Ju96][Khe96][Sa198]. These analogs, present as asmall fraction of the total nucleotide triphosphates, stochastically andirreversibly terminate an elongating DNA chain, because they lack a3′-OH group. Mutant polymerases have improved the uniformity andefficiency of termination, improving the quality of sequencing data[Tab87][Tab95].

While this sequencing strategy has created the “post-genomic world”, ithas well known limitations. Primary among them is that it is difficultto multiplex; each sequence must be determined separately on a separatecapillary. Further, it is not exquisitely sensitive; it cannot determinethe sequence of a small number of molecules, and is insufficientlysensitive to sequence a single molecule of DNA. Further, theirreversibly terminated elongating DNA strands cannot be cloned.Further, the irreversible termination does not introduce a moiety intothe oligonucleotide that can be later used to recover the productstrand.

In part to enhance multiplexing, in part for other reasons, sequencingby synthesis without using electrophoresis was proposed as a strategy in1988 [Hym88]. Generically, the strategy involves detecting the identityof each nucleotide at the same time as it is incorporated into thegrowing strand of DNA in a polymerase-catalyzed reaction. A variety ofarchitectures have been proposed for performing “sequencing bysynthesis” [Che94][Met94]. These differ in the way that the nature ofthe nucleotide that was just incorporated in each step of the synthesisis determined. They also differ by the tactic used to prevent theaddition of the following nucleotide until the identity of thenucleotide that was just incorporated had been determined.

For example, in the pyrosequencing architecture [Ron98], a “minus”strategy is used to look at single nucleotide incorporations. Here, onlyone of the four natural nucleoside triphosphates is incubated in thereaction at any one time. Detection is based on the release ofpyrophosphate during the DNA polymerase reaction, indicating theaddition to the elongating chain of the added triphosphate, or theabsence of the release of pyrophosphate. The pyrophosphate is detectedthrough its conversion to adenosine triphosphate (ATP) by sulfurylase,which then generates visible light in the presence of fireflyluciferase.

The limitations of this procedure are also well known in the art. First,the amount of pyrophosphate must be quantitated to distinguish betweenthe addition of a single nucleotide of the type added, or of several ina “homosequence run”. While this is readily done for runs of one, two,or three nucleotides, it becomes progressively more difficult as theruns become longer. Further, each of the four nucleoside triphosphatesmust be added separately. Polymerases are well known to misincorporatewhen they are not presented with the complementing triphosphate. Thiscreates undesired termination, in many cases, or “ragged ends” inothers.

Another architecture uses a polymerase to direct the incorporation, in atemplate-directed polymerization step, of a nucleoside triphosphate orthiotriphosphate (which is useful in certain architectures) having its3′-hydroxyl group blocked by a removable protecting (or blocking) group.This blocking group prevents the polymerase from adding additionalnucleotides until the blocking group is removed. In practice, thisprovides an arbitrarily long time to determine the nature of the addednucleotide.

A frequent proposal for this architecture is to place differentdistinctive tags on the four nucleobases. These tags may bedistinctively colored fluorescent groups, although other tags have beenproposed. Then, after the blocked nucleotide is incorporated, the natureof the nucleotide incorporated is determined by reading the fluorescencethat comes from the tag. After this is done, the 3′-protecting group isremoved to generate a 3′-OH group at the 3′-end of the elongatingprimer, the tag is removed, and the next cycle of sequencing isinitiated. In this architecture, template-directed polymerization isdone using a DNA polymerase or, conceivably, a reverse transcriptase[Mit03].

When the output is fluorescence, this implementation of the strategyrequires:

-   (a) Four analogues of dATP, dTTP, dGTP, and dCTP, each carrying a    fluorescent dye with a different color, with the 3′-end blocked so    that elongation is not possible.-   (b) The four analogues must be efficiently incorporated, to allow    the elongation reaction to be completed before undesired reactions    occur, and to avoid ragged ends arising from incomplete    incorporation. For single molecule sequencing, failure to    incorporate is still undesirable, as a cycle of sequence collection    is missed.-   (c) The incorporation must be faithful. Mismatched incorporation, if    not corrected by proofreading, will lead to the loss of strands if    the polymerase does not extend efficiently a terminal mismatch. This    will gradually erode the intensity of the signal, and may generate    “out of phase” signals that confuse the reading of the output    downstream. Large numbers of errors will, of course, confuse the    primary signal. For single molecule sequencing, misincorporation may    well mean the end of a read.-   (d) The dye and the group blocking the 3′-OH group need to be    removed with high yield to allow the incorporation of the next    nucleotide of the next nucleotide to proceed. Less than 99%    completion for each cycle (and incompletion) will gradually erode    the intensity of the signal, and may generate “out of phase” signals    that confuse the reading of the output downstream. For single    molecule sequencing, failure to cleave the 3′-OH blocking group may    not create a decisive error, but it can lose a cycle of sequence    data collection.-   (e) The growing strand of DNA should survive the washing, detecting    and cleaving processes. While reannealing is possible, we preferably    would like conditions that allow the DNA primer and template to    remain annealed.

It their most ambitious forms, sequencing-by-synthesis architectureswould use the same nucleoside modification to block the 3′-end of theDNA and to introduce the fluorescent tag [Wel99]. For example, if afluorescent tag is attached to the 3′-position via an ester linkage,replacing the hydrogen atom of the 3′-OH group of the nucleosidetriphosphate, extension following incorporation would not be possible(there is no free 3′-OH group). This would give time to read the colorof the fluorescent label, determining the nature of the nucleotideadded. Then, the 3′-O acyl group could be removed by treatment with amild nucleophile (such as hydroxylamine) under mild conditions (pH<10)to regenerate a free 3′-hydroxyl group, preparing the DNA for the nextcycle.

The difficulty in implementing this elegant approach is the polymerasesthemselves. Any tag that fluoresces in a useful region of theelectromagnetic spectrum must be large, on the order of 1 nm. Crystalstructures of polymerases show that the 3′-position in the deoxyriboseunit is close to amino acid residues in the active site of thepolymerase, and do not offer the incoming triphosphate the space toaccommodate a tag of that size. The structure of the ternary complexesof rat DNA polymerase beta, a DNA template-primer, and dideoxycytidinetriphosphate (ddCTP) from the Kraut laboratory, as well as a variety ofstructures for other polymerases from other sources solved in otherlaboratories, illustrates this fact. The polymerase, therefore, is notlikely to be able to handle substituents having a tag of this size atthe 3′-position. Indeed, polymerases do not work well with anymodification of the 3′-OH group of the incoming triphosphate. Forexample, to accept even 2′,3′-dideoxynucleoside analogues (where the3′-moiety is smaller than in the natural nucleoside), mutatedpolymerases are often beneficial.

Ju et al., in U.S. Pat. No. 6,664,079, noted these problems as theyoutlined a proposal for sequencing by synthesis based on 3′-OH blockinggroups. Therefore, they argued that the prior art had not been enabled,even though it specified many details of an architecture for sequencingby synthesis. They suggested that this problem might be addressed usingnucleotide analogues where the tag, such as a fluorescent dye or a masstag, is linked through a cleavable linker to the nucleotide base or ananalogue of the nucleotide base, such as to the 5-position of thepyrimidines (T and C) and to the 7-position of the purines (G and A).Bulky substituents are known to be accepted at this position; indeed,these are the sites that carry the fluorescent tags in classical dideoxysequencing. According to Ju et al., tags at this position should, inprinciple, allow the 3′-OH group to be blocked by a cleavable moietythat is small enough to be accepted by DNA polymerases. In thisarchitecture, multiple cleavage steps might be required to remove boththe tag (to make the system clean for the addition of the next tag) andthe 3′-blocking group, to permit the next cycle of extension to occur[Mit03][Seo04].

U.S. Pat. No. 6,664,079 then struggled to find a small chemical groupthat might be accepted by polymerases, and could be removed underconditions that were not so harsh as to destroy the DNA being sequencedor the architecture supporting the sequences. U.S. Pat. No. 6,664,079cited a literature report that 3′-O-methoxy-deoxynucleotides are goodsubstrates for several polymerases [Axe78]. It noted, correctly, thatthe conditions for removing a 3′-O methyl group were too stringent topermit this blocking group from being removed under any conditions thatwere likely to leave the DNA being sequenced, or the primer that wasbeing used, largely intact.

An ester group was also discussed as a way to cap the 3′-OH group of thenucleotide. U.S. Pat. No. 6,664,079 discarded this blocking group basedon a report that esters are cleaved in the active site in DNA polymerase[Can95]. It should be noted that this report is questionable, andconsiders only a single polymerase. Therefore, in a modification notconsidered by Ju et al. a formyl group may be used in this architecture.The 3′-O formylated 2′-deoxynucleoside triphosphates are preparable asintermediates in the Ludwig-Eckstein triphosphate synthesis, if the 3′-Oacetyl group that is traditionally used is replaced by a formyl group,and the final alkaline deprotection step is omitted.

U.S. Pat. No. 6,664,079 then cited a literature report that3′-O-allyl-dATP is incorporated by Vent (exo-) DNA polymerase in thegrowing strand of DNA [Met94]. U.S. Pat. No. 6,664,079 noted that thisgroup, and the methoxymethyl MOM group, having a similar size, might beused to cap the 3′-OH group in a sequencing-during-synthesis format.This patent noted that these groups can be cleaved chemically usingtransition metal reagents [Ire86][Kam99], or through acidic reagents(for the MOM group).

These suggestions therefore define the invention proposed in U.S. Pat.No. 6,664,079. Briefly, the essence of that invention is an architecturewhere the triphosphates of four nucleotide analogues, each labeled witha distinctive cleavable tag attached to the nucleobase, and each havingthe hydrogen of the 3′-OH group capped replaced by an allyl group or aMOM group, are used as the triphosphates in the sequencing by synthesisarchitecture, and the products are oligonucleotides prepared bypolymerase incorporation that have this replacement.

This architecture, to date, has never been reduced to practice. This isagain because of the polymerases. While the allyl group is small, todate, no polymerases have been shown to incorporate these to the extentand with the efficiency needed to effectively reduce this invention topractice. Therefore, U.S. Pat. No. 6,664,079 cannot be said to haveenabled the sequencing-by-synthesis strategy. Further, more recentliterature has described the use of Therminator variants to incorporatethese MOM- and allyl-protected nucleoside triphosphates. Therminator hasmany disadvantages that make it difficult to apply in practice. Not theleast of these is the affinity with which it binds to template-primeroverhangs and single stranded DNA, an affinity that makes it difficultto wash in repetitive sequencing-during-synthesis architectures.

Recent patent applications have disclosed a smaller 3′-blocking group,one that has fewer than three heavy (that is, non-hydrogen) atoms. Theirdisclosures taught that such a blocking group is useful for an efficientsequencing-during-synthesis architecture, either with naturalpolymerases or with polymerases in which one of the amino acids incontact with the ribose ring is mutated. These might include the formylunit, where the hydrogen atom of the 3′-OH group is replaced by a COHunit.

In the disclosed invention, the preferred replacement is NH₂ or NHR.There, the 3′-O-amino group is used as a removable protecting group forthe sequencing-by-synthesis scheme. The 3′-O-amino group is chosen is assmall a moiety that forms a stable 3′-O blocking group. The small sizeof the 3′-modification makes it most likely to be accepted by DNApolymerases during template-directed DNA polymerization [Hen04].

Further, contact by DNA polymerases with the 3′-end of the incomingtriphosphate is frequently made with an amino acid with an aromatic sidechain (Phe or Tyr) [Gar99]. The size of this can be reduced (to His),generating the possibility that if any particular natural polymerasedoes not work, then these can be mutated, followed by a round of invitro directed evolution [Gha01], to generate polymerases that accept3′-O-amino triphosphates with acceptable specifications.

The hydroxylamine functionality is stable in water, and displays severalother advantages:

-   (a) 3′-O-Amino-2′-deoxynucleosides [DeC90][Kon85][Bur94][Coo94] are    directly synthesizable from the xylo-2′-deoxyribonucleosides via a    Mitsunobu reaction with N-hydroxyphthalimide.-   (b) The 3′-O-amino-2′-deoxynucleoside blocking group is small, even    smaller than the speculative —OSH unit (which is considered in the    instant invention) and the azido unit (which is incorporated by    reverse transcriptases when they accept azidothymidine triphosphate,    for example).-   (c) The 3′-O-amino-2′-deoxynucleoside functionality has much of the    hydrogen bonding potential of the 3′-OH group. While not wishing to    be bound by theory, these derivatives may form a network of hydrogen    bonds to the catalytic magnesium ion, as suggested by    crystallography for the natural substrate, and therefore fitting    into the active site of various polymerases.-   (d) In some cases, a polymerase can be improved by replacing the Phe    or Tyr (depending on the polymerase) [Eva00][Gar99] that blocks the    3′-position of the incoming triphosphate with a slightly smaller    aromatic and/or hydrophobic group, His/Phe/Val.-   (e) A large number of reagents are known that cleave the N—O linkage    in hydroxylamines and O-alkoxyamines. These are discussed in greater    detail below. Oxidative conditions are provided by bleach, nitrous    acid at pH 6 under conditions where the nucleobases are not    significantly modified, nitroso compounds, iodate, or potassium    ferrate in 1 M NaCl, 50 mM potassium phosphate buffer, 25° C.; this    generates the free —OH group and N₂O, which is trapped. Reducing    agents include catalytic hydrogenation. The preferred approaches    include addition-elimination cycles where the amino group of the    alkoxyamine adds to an electrophile (such as maleimide or a    naphthoquinone) and then ejects the alcohol as a leaving group.-   (f) Once incorporated, the product    3′-O-amino-oligo-2′-deoxyribonucleosides themselves have value,    through capture architectures that exploit the 3′-blocking group or,    after its removal, as the starting point for cloning and further    elongation processes.

With this 3′-O blocking group, other features of the architecture of thestate-of-the-art sequencing-by-synthesis approach can be adopted. Inparticular, the linkers that hold the fluorescent labels to thenucleobases in the Ju architecture might be cleaved using the samereagent is used to remove the amino group from the terminal3′-O-amino-2′-deoxynucleoside.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence of wild-type DNA Polymerase from Thermusaquaticus. Underlined sites represent positions hosting amino acidreplacements identified by the process disclosed here.

FIG. 2. An example multiple sequence alignment between the amino acidsequence of the wild-type DNA polymerase from Thermus aquaticus andsequences from homologs of Taq polymerase. This represents a partiallist of Taq homologs. Taq sites are listed for positions 451-830 onlysince the replacements identified by the process disclosed here arelocated in this domain of the protein. DNA polymerase sequenceshomologous to Thermus aquaticus (THEAQ) are listed for Homo sapiens(HUMAN), Saccharomyces cerevisiae (YEAST), Deinococcus radiodurans(DEIRA), Escherichia coli (ECOLI), Haemophilus influenzae (HAEIN),Bacillus stearothermophilus (BACST), Streptococcus pneumoniae strain R6(STRR6), Mycobacterium tuberculosis (MYCTU), Bacillus phage SPO1 (BPSP1), Mycobacterium phage L5 (BPML5), Bacteriophage T7 (BPT7). Underlinedsites in the Thermus aquaticus (THEAQ) sequence represent positionshosting amino acid replacements identified by the process disclosedhere. Gaps are represented with a period.

DETAILED DESCRIPTION OF THE INVENTION

Engineering DNA Polymerases

The DNA polymerase from Thermus aquaticus (Taq) is commonly used forSanger-based sequencing methods and it is known to releaseprimer/template complexes. Unfortunately, wild-type Taq polymerase maynot be able to incorporate T-ONH₂ well enough to support thesequencing-by-synthesis method outlined above, or other versions of it.Polymerases that have had amino acid replacement(s) (one or more) sothat the variant polymerase (defined here to be different from theparent polymerase, which may be the same or different from a polymerasefound in nature) that accept triphosphates modified to carry reversiblyterminating units (on the sugars as well as elsewhere, such as on thenucleobases) will therefore have utility.

A process to construct useful variants, or the novel compositions thatthose variants are, must begin with a process that identifies sites in apolymerase protein sequence where the amino acid is changed. A “site”is, as defined here”, specified by a number in a sequence of aminoacids, where that number is defined by reference to a figure that liststhe amino acids of the protein in that sequence. Disclosed elsewhere(e.g. U.S. Pat. No. 5,958,784) is a semi-rational approach, defined hereas a “phylogenetic-based approach”, which begins by the process ofanalyzing the patterns of change and conservation among a set ofhomologous proteins. This process comprises some or all of the followingsteps: constructing evolutionary trees from the multiple sequencealignment, inferring the sequences of ancestral proteins at nodes in thetree, applying phylogenetic tools to identify signals/sites associatedwith functional divergence, and examining these sites together with amodel representing the three dimensional structure of the protein. Thisapproach identifies a small number of sites where amino acid replacementmight yield a useful protein, specifically, one that is notcatalytically active or that does not fold properly. Thus, this processcomplements other directed evolution approached.

To apply this process to DNA polymerases that are members ofevolutionary family A, the sequences of 719 polymerases from Family Awere collected from the PFAM database (PF00476) [Bat04]. A phylogenetictree representing the evolutionary relationship of these polymerases wasobtained from the PFAM database (PF00476) [Bat04]. Metrics used to inferfunctional divergence were applied to the dataset. This computationalphylogenetic-based approach confirmed what was already proposed in theliterature: functional divergence of polymerase behaviors has occurredalong branches of the phylogeny separating viral and non-viralpolymerases [Hor95][Lea06][Sis06][Tab95]. Owing to the fact that viralpolymerases are inherently more likely to accept modified nucleotidesthan non-viral polymerases, the phylogenetic-based approach implied thatsites identified as being responsible for functional divergence betweenviral and non-viral polymerases would be sites that, in the non-viralpolymerases, could have their amino acids replaced to generate newpolymerase variants with useful properties.

Making reference to a model for the three dimensional crystal structureof polymerases, phylogenetic-based analysis identified sites both withinand without the active-site cleft of the polymerase. Rationally, siteswithin the active site are more likely to alter substrate specificity[Hen05]. Amino acid replacements distributed across 35 of these siteswere identified as having potential interest. According to the processof the invention, one of these sites may be changed, or more than one ofthese sites may be changed, to generate a useful polymerase. Further,these sites may be changed starting with a polymerase parent from anymember of Family A (either as found naturally or as found from alaboratory that has already one or more amino acids different from anatural Family A polymerase.

Example 1 Useful, Novel Variants of Taq Polymerase

The Taq polymerase protein is widely used for sequencing DNA. Standardexperimental approaches are used to perform site-directed mutagenesis toincorporate the amino acid replacements at sites listed in the Claims.For instance, mutagenic PCR is performed on a template Family Apolymerase gene using the appropriate mutagenic primers to generatevariants containing amino acid replacements. The PCR mixtures containthe following: 1× Mutagenic Taq Buffer (10 mM Tris-HCl, pH 8.3, 50 mMKCl, 15 mM MgCl₂), 0.1 ng/μL template DNA, 200 μM dNTPs, 300 nM P-4, 300nM mutagenic primers, 5 U Taq polymerase (New England BioLabs, Beverly,Mass.), and MgCl₂. PCR reaction continues as follows: 5 min, 94° C.; (30s, 94.0° C.; 20 s, 55.0° C.; 3 min, 72.0° C.)×15 cycles; 7 min, 72.0°C.; 4.0° C. Products can purified with the QIAquick PCR Purification Kit(Qiagen, Valencia, Calif.), eluted with Qiagen Buffer EB (50 μL), andquantitated at an absorbance of 260 nm using a Spectrophotometer.

This invention provides new sequences of proteins that are likely toaccept nucleoside 3′-ONH₂ blocked triphosphates, dideoxynucleosidetriphosphates and C-glycosides such as2′-deoxypseudouridine-5′-triphosphate.

REFERENCES

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What is claimed is:
 1. A DNA polymerase from Thermus aquaticus whereinthe amino acid at position 616, as defined by SEQ ID NO:1, is replacedby an amino acid selected from the group consisting of Ala, Arg, Cys,Glu, Gln, Gly, His, Ile, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val and Asp.